JP3824312B2 - Dried bacterial cells by spray drying - Google Patents

Description

表１に示す通り、本発明に係る噴霧乾燥を用いれば、凍結乾燥を用いるよりも生存率の高い菌体乾燥物が得られることがわかった。
【００６２】
〔菌体乾燥物の粒径〕
実施例１において、入り口温度を６０℃として得られた本発明にかかる菌体乾燥物を、顕微鏡（株式会社キーエンス製、デジタルＨＤマイクロスコープＶＨ−７０００）を用いて測定した。粒径の分布は、以下の様であった。
【表２】

【００６３】
〔実施例２〕
実施例１と同様にして、菌体を培養し、菌懸濁液を作成し、菌懸濁液中の生菌数の測定した。
〔噴霧造粒〕
流動層造粒機ＦＬＯ−１２０（フロイント社製）に、従来の２流路の噴霧ノズルに加えてさらに４流路ノズル（特許２７９７０８０に記載のもの）を組み込んだ装置を用いて、流動層中に乳酸菌懸濁液を噴霧することにより乳酸菌含有粉末を作成した。
工程は大きく二つに分けられ、第一工程は２流路ノズルを用いて粒状物としての賦形剤に結合剤を吹き付け、造粒を行った。第二工程では、引き続き流動層を低温で維持した状態で４流路ノズルを用い乳酸菌懸濁液を噴霧乾燥すると同時に第一工程で得られた粒状物と混合した。
本実験で使用された機械の運転条件は、給気風量が７０ｍ^３／分、ノズルエア量が４０ＮＬ／分（２０℃で換算した値）であった。
また、造粒に用いられた賦形剤、結合剤の組成は以下のものを用いた。

【００６４】
第一工程について以下に詳細に述べる。上記賦形原料１２０ｋｇを秤量しホッパーに入れ、給気温度を１００℃に設定し乾燥空気を送り込み流動層を形成させ、２流路ノズルを用いて結合剤を液流速１．６Ｌ／分で噴霧した。この時の排気温度は４５〜５０℃、流動層内温度は５５〜６０℃であった。結合剤の噴霧が終了すれば、そのまま流動層で２０分間乾燥させて低水分の造粒物を得た。
【００６５】
次に、第二工程について以下に詳細に述べる。造粒物の後乾燥終了後、給気温度を７０℃に下げた状態で４流路ノズルを用いて実施例１と同様にして作成した菌懸濁液を液流速１６ｍＬ／分で３５０ｍＬ噴霧した。この時の排気温度は３０〜３５℃、流動層内温度は３５〜４０℃であった。このような低温での噴霧乾燥により乳酸菌は粉末化され、同時に流動層内で混合することにより本発明に係る乳酸菌含有粉末を得た。
【００６６】
〔比較例２〕
製剤中に乳酸菌が同量含まれるように調整した乳酸菌混合粉末を作成した。
実施例１で得られた菌体粉末２ｇをトウモロコシデンプンで１０倍に倍散して２０ｇとし、実施例２の第一工程で得られた賦形剤と結合剤とからなる造粒物１１８０ｇに加え、Ｖ型混合機で１００ｒｐｍ、３分間混合したものを乳酸菌混合粉末とした。
【００６７】
実施例２または比較例２で得られた粉末１ｇ中に含まれる生菌数を、実施例１の〔菌体乾燥物中の生菌数の測定〕の項にしたがって測定した。実施例２では生菌数は５．１２×１０^８（ＣＦＵ／ｇ）であり、比較例では生菌数は４．８５×１０^８（ＣＦＵ／ｇ）であった。
したがって、噴霧乾燥と流動層造粒を組み合わせた本発明に係る方法を用いれば、従来の噴霧乾燥、造粒および混合工程よりなる粉末の製造方法と少なくとも同程度、あるいはそれ以上の生菌率を保ちつつ、製造工程の簡略化が可能であることがわかった。
【００６８】
〔実施例３〕
蒸留水５００ｇに、下記成分を添加し、攪拌してコーティング剤を作成した。

一方、実施例１と同様にして、菌懸濁液を作成した。
４流路ノズル（特許２７９７０８０に記載のもの）を組み込んだ図２に示した噴霧乾燥機を用いて噴霧乾燥した。すなわち、２本の液体流路と２本の気体流路を有する４流路ノズルにおける一の液体流路から菌体液を５ｇ／分の速度で供給し、他の液体流路から液状のコーティング剤を２０ｇ／分の速度で供給し、２本の気体流路から圧縮気体を４０ＮＬ／分の速度で供給して生じた噴霧液滴を、乾燥風により乾燥し、本発明に係る部分コーティング菌体乾燥物を製造した。
なお、乾燥風は、入り口温度が１００℃であり、１．０ｍ^３／分の割合で０．９ｍＰａの乾燥室内に供給した。
従来のビフィズス菌原末と本実施例にかかる部分コーティング菌体乾燥物をそれぞれ１０ｍｇとり、食味試験を行ったところ、本実施例にかかる部分コーティング菌体乾燥物の食味は改善されていた。
【００６９】
〔実施例４〕
実施例１と同様に作成した菌懸濁液１００ｇと下記組成の腸溶性コーティング基剤を混合し、該混合液を４流路ノズル（特許２７９７０８０に記載のもの）を組み込んだ図２に示した噴霧乾燥機を用いて噴霧乾燥した。すなわち、２本の液体流路から混合液を５．６ｇ／分の速度で供給し、２本の気体流路から圧縮気体を４０ＮＬ／分の速度で供給して生じた噴霧液滴を、乾燥風により乾燥し、本発明にかかる腸溶性コーティングされた菌体乾燥物を製造した。
なお、乾燥風は、入り口温度が８０℃であり、１．０ｍ^３／分の割合で０．９ｍＰａの乾燥室内に供給した。

【００７０】
〔腸溶性コーティングされた菌体乾燥物の耐酸性試験〕
日本薬局方・一般試験法・崩壊試験法に記載の第１液（ｐH１．２、３６〜３８℃）を用いて耐酸性試験を行った。１０００ｍＬ程度の保温できる容器に第１液を入れ３６〜３８℃に保温した。これに得られた菌体乾燥物を加えて良く攪拌し、菌体乾燥物投入５、１５、３０、６０分後に１ｍLずつサンプリングした。サンプリングした献体は、実施例１に記載した希釈液を加えて適当な濃度に順次希釈し、実施例１と同様にして生菌数の測定を行った。なお、局方第１液は、人工胃液であり、塩化ナトリウム２．０ｇに塩酸７ｍLおよび蒸留水を加えて溶かし１０００ｍLをしたものである。
また、従来のビフィズス菌原末についても、同様の試験を行った。
図３に第１液保護時間と生菌数の関係を示した。従来の菌原末においては投入５分後で生菌数が１０００分の１以下となり、３０分後以降は生菌が認められなくなったのに対して、本発明にかかるコーティング菌原末においては投入６０分後においても１％程度の生菌が認められた。つまり、上記のコーティングを行うことで菌体乾燥物の耐酸性が大幅に改善された。
【００７１】
〔実施例５〕
菌体としてLactobacillus acidophilusを用いた以外は、実施例１と同様にして菌懸濁液を作成した。
菌懸濁液中の生菌数を日本薬局方外医薬品規格「ラクミトン」の項に記載されているラクミトンの定量法に準じて測定した。すなわち、菌懸濁液１ｍｌをとり、下記のようにして調製した希釈液を用いて１０倍希釈法にて１ｍｌ中の生菌数が２０〜２００個となるよう希釈した。この液１ｍｌをペトリ皿にとり、ここに５０℃に保ったラクミトン試験用カンテン培地を２０ｍｌ加えてすばやく混和し、固化させた。これを３７℃で４８時間培養し、出現したコロニー数と希釈倍率から菌懸濁液中の生菌数を求めた。
【００７２】
ここで、ラクミトン試験用カンテン培地は、下記成分を混合し、高圧蒸気滅菌器を用いて１２１℃で１５分間加熱して滅菌し作製した。
ここで、トマトジュースは、トマトジュースに等量の精製水を加え時々かき混ぜながら煮沸した後、ｐＨを６．８に調整しろ過したものを用いた。
カゼイン製ペプトン ２０ｇ
酵母エキス ５ｇ
肉エキス １５ｇ
ブドウ糖 ２０ｇ
Ｌ−塩酸システイン １ｇ
ポリソルベート８０ ３ｇ
トマトジュース ２００ｍＬ
カンテン ２０ｇ
精製水 ８００ｍＬ
ｐＨ ６．８
【００７３】
上記希釈液は、下記成分を混合し、高圧蒸気滅菌器を用いて１２１℃で１５分間加熱して滅菌し、調製した。
リン酸一水素ナトリウム ６．０ｇ
リン酸二水素カリウム ４．５ｇ
ポリソルベート８０ ０．５ｇ
Ｌ−塩酸システイン ０．５ｇ
カンテン １．０ｇ
精製水 １０００ｍｌ
ｐＨ ６．９
【００７４】
上記にように作成した菌懸濁液を、実施例１と同様に噴霧乾燥した。
機械の運転条件は、入り口温度が６０℃、排気温度が４５℃であった以外は実施例１と同様であった。
得られた菌体乾燥物中の生菌数を日本薬局方外医薬品規格「ラクミトン」の項に記載されているラクミトンの定量法に準じて測定した。すなわち、菌体乾燥物５gをあらかじめ３７℃に加温した希釈液にとり、全量を５０ｍＬとした。これをよく攪拌した後３７℃で３０分間加温し、試料原液とした。これを希釈液を用いて１０倍希釈法にて１ｍＬ中の生菌数が２０〜２００個となるよう希釈した。この液１ｍＬをペトリ皿にとり、ここに５０℃に保ったラクミトン試験用カンテン培地を２０ｍＬ加えてすばやく混和し、固化させた。これを３７℃で４８時間嫌気培養し、出現したコロニー数と希釈倍率から菌体乾燥物中の生菌数を求めた。
【００７５】
菌体乾燥物中の生菌数は７．２４×１０^１１（ＣＦＵ／ｇ）であり、生存率は９０％であった。なお、生存率は実施例１と同様にして求めた。
したがって、本発明にかかる噴霧乾燥を用いれば菌体の生存率が高まり、その結果生菌数の非常に多い菌体乾燥物が得られることがわかった。
【００７６】
【発明の効果】
シングルミクロンの噴霧液滴を形成できる微粒化装置を備えた噴霧乾燥装置を用いることにより、噴霧液滴の単位重量あたりの表面積が大きくなり、乾燥風との接触が効率よく行われる。したがって、従来の噴霧乾燥と同程度の温度の乾燥風より乾燥させた場合、菌体液の乾燥に要する時間が短くなり、菌体の死滅または損傷を極力抑えることができる。また、乾燥風の温度を従来の噴霧乾燥における乾燥風の温度より低くしても、収量または安定性を落とすことなく十分な乾燥を行うことができ、また乾燥風の温度が低いので乾燥風の熱による菌体の死滅または損傷を極力抑えることができる。したがって、本発明にかかる菌体乾燥物の製造方法により、生菌数の多い菌体乾燥物を得ることができる。
【００７７】
また、このように粒径の小さな噴霧液滴を乾燥風で乾燥することによって得られるシングルミクロンサイズの粒子を含有する菌体乾燥物は、圧縮成形等の際に菌体にかかる圧力が少なくて済み、また静電気も起こりにくいなど、錠剤などの剤形の医薬または食品に成形しやすい。
【００７８】
本発明にかかる噴霧乾燥装置として、２本の液体流路と２本の気体流路を有する４流路ノズルを有する噴霧乾燥装置を用いることにより、液体流路が２本あるので大量噴霧が可能になり製造効率が上がるほか、２種の異なる溶液または懸濁液を別々の液体流路から同時に噴霧することにより、これらが混合された菌体乾燥物を製造できる。
また、一方の液体流路からは液状のマスキング剤もしくはコーティング剤を、他方の液体流路からは菌体液を噴霧することにより、マスキングもしくはコーティングされている菌体乾燥物を製造できる。したがって、マスキングもしくはコーティングを別途行う必要がなくなるので製造工程が少なくなる。また、同様にしてカプセル基材を菌体液と同時に噴霧することによってマイクロカプセルを製造することもできる。
さらに、４流路ノズルを有する噴霧乾燥装置を用いることにより、粒度分布が比較的狭い範囲に収まるため、その後の整粒、篩過等の工程を行いやすい。
【００７９】
噴霧乾燥は、装置の規模が凍結乾燥の場合に比べて小規模であり、かつ装置の運転に要するエネルギーも少ないことから、コスト的にも資源的にも有用である。
また、本発明にかかる粉状物と結合剤を用いて流動層造粒を行う工程と、噴霧乾燥によりシングルミクロンの菌体乾燥物を得る工程とを、同一容器内で行うことを特徴とする装置を用いて、該装置内で造粒、菌体の噴霧乾燥、混合を連続的に行うことにより、製造工程が簡略化できると同時に、菌体乾燥物と造粒物の混合均一性が向上し分級も起こりにくくなる。
【図面の簡単な説明】
【図１】４流路ノズルを有する噴霧乾燥装置におけるノズルエッジ部分の内部構造を示す。
【図２】４流路ノズルを有する噴霧乾燥装置の概要図を示す。
【図３】腸溶性コーティングされた菌体乾燥物の耐酸性試験における生菌数の経時的変化を示す。
【図４】本発明にかかる粉状物と結合剤を用いて流動層造粒を行う工程と、噴霧乾燥によりシングルミクロンの菌体乾燥物を得る工程とを、同一容器内で行うことを特徴とする装置の概要図を示す。
【符号の説明】
１、２ 圧縮気体が供給される気体流路
３、４ 被乾燥体を含む液体が供給される液体流路
５ 流体流動面
６ 衝突焦点
７ 噴霧液滴
２１ 菌体液、コーティング剤またはマスキング剤
２２ 液体流路
２３ 気体流路
２４ 圧縮空気送入口
２５ 乾燥風送入口
２６ 入り口温度計
２７ 微粒化装置
２８ 噴霧ノズル
２９ 出口温度計
３０ 乾燥室内圧力計
３１ 乾燥室
３２ 菌体乾燥物出口
３３ 整流器
４１ 装置本体
４２ 流動床
４３ バグフィルター
４４ 菌体液
４５ 結合剤
４６ 菌体液噴霧用ノズル
４７ 結合剤噴霧用ノズル
４８ 粉状体
４９ 流動用気体[0001]
[Technical field to which the invention belongs]
The present invention relates to a method for producing a dried microbial cell product using spray drying, a dried microbial cell product obtained by the production method, and a composition such as a medicine or food containing the dried microbial cell product.
[0002]
[Prior art]
As a drying method in the production process of a medicine or food, freeze drying or vacuum freeze drying is frequently used. Freeze drying is a method of drying at a low temperature of about −20 ° C. to −160 ° C. using liquid nitrogen or the like, and vacuum freeze drying is a method of drying at a pressure of about 35 ° C. or less and about 50 to 400 hPa. It is.
[0003]
However, when freeze-drying or vacuum freeze-drying is adopted as a drying method in the production process of lactic acid bacteria, yeast, and other fungus bodies, or pharmaceutical or food containing the same, the fungus bodies are killed or damaged during drying. There was a drawback of receiving. This is because it takes a long time to reach the freezing temperature in freeze-drying or vacuum freeze-drying, and the bacteria are damaged by the metabolites of the bacteria generated during that time and the freezing process. In particular, the vacuum freeze-drying has a very slow drying rate, and if the moisture in the medicine or food is all removed, the drying time becomes longer, so the above problem becomes more remarkable.
[0004]
Moreover, in order to perform freeze-drying or vacuum freeze-drying, a very large-scale installation is required, and the capital investment is also very expensive.
Furthermore, when freeze-drying or vacuum freeze-drying is employed as a drying method, it becomes difficult to manufacture by a continuous process, and thus the manufacturing process becomes complicated. Further, after the freeze-drying or vacuum freeze-drying, further steps such as pulverization and homogenization are required, so that the number of production steps increases, and the killing of bacteria during that time also becomes a problem.
Such problems have been pointed out regarding freeze-drying or vacuum freeze-drying.
[0005]
On the other hand, as a drying method, spray drying is known in addition to freeze drying or vacuum freeze drying. Spray drying is a method in which a liquid is made into droplets by a atomizer and moisture is evaporated and dried by bringing the droplets into contact with a relatively high temperature drying air.
However, conventional spray drying has been considered to be generally unsuitable for the production of pharmaceuticals or foods containing viable bacteria because the temperature of the drying air is high and the cells are killed by the heat of the drying air. Further, when the temperature of the drying air is lowered to solve this problem, there is a drawback that a sufficiently dried microbial cell product cannot be obtained and both yield and stability are lowered.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for drying a bacterial cell liquid capable of suppressing the death or damage of the bacterial cells as much as possible and maintaining a high viable cell ratio in the production process of the dried bacterial cell product or a composition such as a pharmaceutical or food containing the same. There is to do.
Another object of the present invention is to provide a single micron-sized dried microbial cell product that has a large number of microbial cells per unit weight and is easy to mold as a medicine or food. Still another object of the present invention is to provide a method for producing a bacterial cell composition that can greatly simplify the production process and an apparatus according to the production method.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have obtained the knowledge that problems with conventional spray drying can be solved by drying single micron spray droplets with drying air.
That is, the spray droplets formed in the conventional spray drying apparatus are about 10 to 40 μm, but when this is a very small spray droplet of a single micron, the surface area per unit weight of the spray droplet is reduced. Since it becomes large, the contact with dry wind is performed efficiently. Therefore, when drying with drying air at a temperature similar to that of conventional spray drying, the time required for drying the bacterial cell liquid is shortened, and killing or damage of the bacterial cells can be suppressed as much as possible. Even if the temperature of the drying air is lower than the temperature of the drying air in the conventional spray drying, sufficient drying can be performed without reducing the yield or stability, and the temperature of the drying air is low. The death or damage of the bacterial cells due to heat can be suppressed as much as possible.
[0008]
Further, the present inventors have found that a dried bacterial cell product obtained by drying a single micron spray droplet with a drying air has a small particle size and contains particles of a single micron size. Dried bacterial cells with a small particle size have the advantage that they can be easily molded into pharmaceuticals or foods in dosage forms such as tablets, as the pressure applied to the cells during compression molding is low and static electricity is less likely to occur. .
[0009]
That is, the present invention
(1) Single micron dried cells
(2) The number of viable bacteria is 1.0 × 10 ⁵ ~ 2.0 × 10 ¹² The dried microbial cell product according to (1) above, which is CFU / g,
(3) The number of viable bacteria is 1.0 × 10 ¹¹ ~ 2.0 × 10 ¹² A dried microbial cell characterized by CFU / g,
(4) Bacterial cells are bifidobacteria, lactobacilli, lactic acid cocci, spore-forming lactic acid bacteria, saccharifying bacteria, yeast, bacilli, actinomycetes, Bacillus toyoi, B. dried cell bodies according to the above (1) to (3), which are licheniformis, butyric acid bacteria, acetic acid bacteria or amino acid fermenting bacteria,
(5) The dried microbial cell product, wherein the dried microbial cell product according to (1) to (4) is further coated or masked,
(6) The method for producing a dried microbial cell product according to the above (1) to (5), wherein the single micron spray droplets are dried with drying air,
(7) The method for producing a dried bacterial cell product according to (6), wherein spray droplets of single micron are formed by a four-channel nozzle,
(8) The method for producing a dried microbial cell product according to (6) or (7) above, wherein the drying air has a temperature of 5 to 150 ° C. at the entrance of the drying chamber,
(9) The bacterial cell according to (5) above, wherein the liquid coating agent or masking agent and the bacterial cell liquid are sprayed simultaneously to form spray droplets, and the spray droplets are dried with dry air. A method for producing a dried product,
(10) Bacterial fluid is supplied from one liquid channel in a four-channel nozzle having two liquid channels and two gas channels, and a liquid coating agent or masking agent is supplied from another liquid channel And the method for producing a dried microbial cell product according to (9) above, wherein the spray droplets produced by supplying compressed gas from two gas flow paths are dried with dry air,
(11) A microbial cell characterized in that a step of performing fluidized bed granulation using a powder and a binder and a drying step of obtaining a single micron microbial cell dried product by spray drying are performed in the same container. Production method of the composition,
(12) An apparatus characterized in that a fluidized bed granulation process using a powder and a binder and a drying process for obtaining a single micron dried product by spray drying are performed in the same container,
(13) A bacterial cell composition characterized in that a dried product of single-micron bacterial cells is attached to a granulated product composed of a powdery material and a binder,
(14) A pharmaceutical product or food containing the dried microbial cell product according to (1) to (5) or the microbial cell composition according to (13), and
(15) Single micron droplets containing bacterial cells,
About.
[0010]
The present invention also provides:
(1) Single micron dried microbial cells obtained by drying single micron spray droplets with dry hot air,
(2) The dried bacterial cell product according to (1), wherein a single micron spray droplet is formed by a four-channel nozzle,
(3) The dried cell body according to (1) or (2), wherein the inlet temperature of the drying hot air in the drying chamber is 5 to 150 ° C.
(4) The dried bacterial cell product according to (1) to (3), which is coated or masked,
(5) The sprayed liquid droplets are formed by spraying a liquid coating agent or masking agent and a bacterial cell liquid at the same time, and the dried bacterial cell product according to (1) to (4) above,
(6) In a four-channel nozzle having two liquid channels and two gas channels, a bacterial cell liquid is supplied from one liquid channel, and a liquid coating agent or masking agent is supplied from another liquid channel. Bacterial drying according to any one of (1) to (5) above, wherein single micron spray droplets generated by supplying and supplying compressed gas from two gas flow paths are dried with dry hot air object,
(7) The cells are bifidobacteria, lactobacilli, lactic acid cocci, spore-forming lactic acid bacteria, saccharifying bacteria, yeast, bacilli, actinomycetes, Bacillus toyoi, B. dried cell bodies according to the above (1) to (6), which are licheniformis, butyric acid bacteria, acetic acid bacteria or amino acid fermenting bacteria,
(8) Viable count is 1.0 × 10 ¹¹ ~ 2.0 × 10 ¹² The dried microbial cell product according to the above (1) to (7), which is CFU / g, and
(9) A medicine or food containing the dried microbial cell product according to (1) to (8) above,
Also related.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The dried microbial cell in the present invention usually refers to a dried individual microbial cell or a collection of dried microbial cells.
As long as the dried microbial cell product according to the present invention or the dried microbial cell product contained in the pharmaceutical or food composition according to the present invention includes a dried microbial cell product having a particle size of single micron, in any case. It belongs to the present invention. Therefore, when the dried microbial cell product is a mixture of a dried microbial cell product having a particle size of a single micron and a dried microbial cell product having a particle size of more than a single micron, for example, the average value of the particle size may be a single micron. It belongs to the present invention.
Here, the single micron means a value of 1 to 10 μm by rounding off the first decimal place.
[0012]
The microbial cells used in the present invention are not particularly limited, and examples thereof include Bifidobacterium such as Bifidobacterium bifidum, B. longum, B. breve, B. adrecentis, B. infantis, B. pseudolongum, and B. thermophirum; for example, Lactobacillus Lactobacilli such as acidophilus, L. casei, L. gasseri, L. plantalum, L. delbrueckii subsp bulgaricus, L. delbrueckii subsp lactis; ) Lactococci such as hirae, Lactococcus lactis, Streptococcus thermophilus; for example, spore-forming lactic acid bacteria such as Bacillus coagulans; Aspergillus oryzae, Asp. Nigar, Asp. Neisseria gonorrhoeae such as Sojae; Actinomycetes such as Streptomyces; for example, Bacillus toyoi, B. et al. Examples include butyric acid bacteria such as licheniformis and Clostridium butyricum; acetic acid bacteria such as Acetobacter; amino acid-fermenting bacteria such as Corynebacterim, and other useful bacteria.
[0013]
The above-mentioned microbial cells can be obtained by culturing under known conditions or similar conditions. For example, in the case of lactic acid bacteria, one or more of the lactic acid bacteria are usually cultured at about 25-45 ° C. for about 4-24 hours in a liquid medium containing glucose, yeast extract, peptone, etc., and cultured. Bacteria are collected from the liquid and washed to obtain wet cells.
The microbial cell may be a processed product of the microbial cell. Examples of the treatment include extraction with an appropriate solvent. Specifically, yeast extract etc. which are the hot water extract of yeast are mentioned, for example.
[0014]
In the method for producing a dried bacterial cell according to the present invention, first, the bacterial cell is dissolved in a solvent to obtain a bacterial cell liquid. As the solvent, a known solvent used in the art may be used, but water is preferable. If desired, ethyl alcohol or the like may be added. By adding ethyl alcohol or the like, ethyl alcohol or the like is first vaporized and then water is vaporized, so that stepwise drying is possible.
The cell fluid may be a suspension. As described above, a known solvent may be used as the solvent, but water or an aqueous solution obtained by adding ethyl alcohol or the like to water is preferable. When suspending, a suspending agent may be used. As the suspending agent, known ones such as sodium alginate or methylcellulose may be used.
[0015]
A protective agent may be added to the bacterial cell solution.
The protective agent may be one commonly used in the art, for example, vitamins such as ascorbic acid; for example, amino acids such as glutamine, glutamic acid, L-cysteine, glycine, phenylalanine, serine or threonine; for example, glucose, fructose Sugars such as sucrose, maltose, mannitol or maltitol or sugar alcohols; for example, polysaccharides such as oligosaccharides, cyclodextrins or dextrins; fats such as higher fatty acids obtained from rapeseed, soybean or peanuts; , Protein obtained from milk or soybean, or a proteolysate such as a peptide; for example, inorganics such as magnesium sulfate; or others such as sucrose fatty acid ester, malic acid, nucleic acids, yeast extract, nonfat dry milk, peptone, gelatin Properly it can be mentioned tannin, and the like.
As the protective agent, the above substances may be used alone or in combination of two or more kinds. Moreover, it is preferable to add this protective agent so that it may become about 0.1-5.0 times amount, preferably about 0.5-3.0 times amount of wet cell weight.
[0016]
Additives commonly used in the art such as excipients, binders, disintegrants or antistatic agents may be added to the above bacterial cell liquid at a usual blending ratio.
Examples of the excipient include sugars such as lactose, granulated sugar or corn starch; sugar alcohols such as mannitol; celluloses such as natural cellulose or crystalline cellulose; cellulose derivatives such as hydroxycellulose and the like. Examples of the crystalline cellulose include water-insoluble cellulose in the form of white powder obtained by finely refining the network molecular structure to some extent by, for example, acid treatment of natural cellulose. Examples thereof include a mixture of crystalline cellulose and carmellose or carmellose sodium.
Examples of the binder include hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, dextrin or pregelatinized starch.
Examples of the disintegrant include a cross-linked product (Akizol) such as carmellose calcium, carmellose or carmellose sodium, a cross-linked product (Prasidone) such as polyvinylpyrrolidone, corn starch or low-substituted hydroxypropyl cellulose.
Examples of the antistatic agent include thyroid, talc, and aerosil.
[0017]
In the method for producing a dried microbial cell product according to the present invention, the microbial cell solution is then subjected to spray drying. As the spray drying apparatus used for the spray drying, a spray drying apparatus equipped with a atomizing apparatus capable of forming single micron spray droplets is preferable. When spray droplets with a very small particle size are used, the surface area per unit weight of the spray droplets is increased, the contact with the drying air is efficiently performed, and the killing or damage of bacteria due to the heat of the drying air is minimized. It is because it can do.
Here, the spray droplet of a single micron means that the particle size of the spray droplet is 1 to 10 μm rounded off to the first decimal place.
[0018]
Specifically, as the spray drying device, for example, a spray drying device in which the atomization device is a rotary atomizer (rotary disk), a pressurized nozzle, or a two-channel nozzle or a four-channel nozzle using the force of compressed gas. Can be mentioned.
In the present invention, any of the above-mentioned spray drying apparatuses may be used regardless of the type, but a spray drying apparatus having a four-channel nozzle is used from the viewpoint of obtaining fine spray droplets. preferable. The spray drying apparatus may be known per se.
[0019]
As the structure of the four-channel nozzle in the spray drying apparatus having the four-channel nozzle, when the gas channel and the liquid channel are one system, two systems are provided symmetrically at the nozzle edge, and the fluid flow surface An inclined surface is provided on the nozzle edge.
One embodiment of the structure of the four-channel nozzle will be described in more detail with reference to FIG. At the nozzle edge of the four-channel nozzle, the bacterial cell liquid that springed out from the liquid channel 3 or 4 was thinly stretched and stretched on the fluid flow surface 5 by the high-speed gas flow exiting from the gas channel 1 or 2. The liquid is atomized by a shock wave generated at a collision focal point 6 at the tip of the nozzle edge to form a single micron spray droplet 7.
As described above, it is preferable that the four-channel nozzle adopts an external mixing method in which the compressed gas and the liquid are gathered at one point from both sides toward the collision focus at the nozzle edge tip. This is because the nozzle can be sprayed for a long time without clogging.
[0020]
As the compressed gas, for example, an inert gas such as air, carbon dioxide, nitrogen gas, or argon gas can be used. In particular, when spray-drying things that are easily oxidized, it is preferable to use an inert gas such as nitrogen gas or argon gas.
The compressed gas pressure is about 1 to 15 kgf / cm. ² Degree, preferably about 3-8 kgf / cm ² Degree.
The amount of gas in the nozzle is about 1 to 100 L / min, preferably about 5 to 50 L / min, more preferably about 10 to 20 L / min per 1 mm of the nozzle edge.
[0021]
One embodiment of a spray drying apparatus having a four-channel nozzle is shown in FIG. When one embodiment of the spray drying according to the present invention is described with reference to FIG. 2, the bacterial cell liquid (reference numeral 21) is sprayed from the four-channel nozzle (reference numeral 28) as described above with reference to FIG. . In the drying chamber (reference numeral 31), the spray droplets are brought into contact with the drying air sent from the drying air inlet (reference numeral 25) through the rectifier (reference numeral 33) to evaporate the water and dry the microbial cells. Get.
The temperature of the drying air at the entrance of the drying chamber (hereinafter referred to as “inlet temperature”) is about 2 to 400 ° C., preferably about 5 to 250 ° C., more preferably about 5 to 150 ° C. Even when the inlet temperature is about 200 to 400 ° C., the temperature of the object to be dried is not so high due to the heat of vaporization due to the evaporation of moisture, and the residence time in the drying chamber is shortened, Death and damage can be minimized.
The temperature of the drying air at the outlet of the drying chamber (hereinafter referred to as “outlet temperature”) is about 0 to 120 ° C., preferably about 5 to 90 ° C., more preferably about 5 to 70 ° C.
In the spray drying apparatus shown in FIG. 2, the inlet temperature refers to the temperature measured by the inlet thermometer (reference numeral 26), and the outlet temperature refers to the temperature measured by the outlet thermometer (reference numeral 29). Say.
[0022]
The drying chamber may be depressurized. By reducing the pressure, there is an advantage that the drying efficiency is increased and the drying time is shortened. The pressure after the pressure reduction in the drying chamber differs depending on the size of the spray droplets and the like. ³ ~ 7.0 × 10 ³ It is mentioned that it is about Pa.
[0023]
If a spray drying apparatus having a four-channel nozzle is used, since there are two liquid channels in the nozzle as described above, two different types of bacterial body fluids or bacterial body fluids and other solutions or suspensions are separated. By spraying simultaneously from these liquid flow paths, it is possible to produce a dried microbial cell product in which these are mixed. For example, by simultaneously spraying two different types of bacterial cell solutions, a dried bacterial cell product containing the two types of bacterial cells can be obtained. Moreover, the microbial cell dried material containing a microbial cell and an edible composition is obtained by spraying a microbial cell liquid and an edible composition simultaneously.
[0024]
Examples of the edible composition used in the present invention include a liquid composition of milk, meat, fish, fruit or vegetable. Or two or more of these may be mixed.
These edible compositions are preferably concentrated to a moisture content of less than about 70% by weight prior to spray drying, while leaving a level of moisture that does not lose fluidity.
[0025]
Examples of the liquid composition of fruits or vegetables include heating of seeds, roots, tubers, stems, leaves, flowers or fruits derived from edible plants or raw finely pulverized parts.
Preferred fruits or vegetables include, for example, leaves such as leek, leek, asparagus, fennel or cabbage; eg, stems such as sardine or broccoli; seeds obtained from eg cocoa, peas, soybeans or cereals; Roots such as onion, radish, celery or beet; for example, tubers such as cassava or potato; Fruits such as sea buckthorn berry quince or mirabelle cherry; or mushrooms.
Examples of the liquid composition of milk, meat or fish include milk, egg, meat or fish; heated or raw fine groin; protein or protein hydrolyzate obtained therefrom.
[0026]
Further, in the spray drying apparatus having a four-channel nozzle, the bacterial cell liquid is sprayed from one of the two liquid channels in the nozzle, and the liquid coating agent or masking agent is sprayed from the other liquid channel. By spraying, the bacterial cells can be coated or masked, and the dried or coated bacterial cell according to the present invention can be obtained.
By coating the cells, the time of disintegration can be adjusted, or it can be adjusted to act on a specific organ such as the intestine. In addition, the appearance and quality can be improved. Furthermore, in a medicine or food containing two or more components, when the components are altered by contact with each other, the alteration can be suppressed by coating either or both components.
Moreover, bitterness and sourness can be suppressed by masking the cells. In particular, a pharmaceutical or food containing a high concentration of lactic acid bacteria or the like has a unique taste, and the unique taste can be alleviated by coating or masking part or all of the surface of the fungal powder. There are advantages.
[0027]
As the coating agent used in the present invention, for example, a known material having a covering ability such as hydroxypropylcellulose (hereinafter abbreviated as HPC), starch paste, sugar solution such as silicon, candy, or various coating base materials is used. Good.
For example, enteric coating substrates include hydroxypropylmethylcellulose phthalate (HPMCP), hydroxypropylmethylcellulose acetate succinate (HPMCAS), cellulose acetate phthalate (CAP), carboxymethylethylcellulose (CMEC), and methacrylic acid-acrylic acid ethyl ester. Examples thereof include a polymer and a methacrylic acid-methacrylic acid methyl ester copolymer. Examples of other coating substrates include ethyl cellulose, hydroxypropylmethyl cellulose, cellulose acetate phthalate, polyvinyl acetal diethylaminoacetate, and the like.
Examples of the masking agent used in the present invention include aminoacryl methacrylate copolymer E, aminoacryl methacrylate copolymer RS, methacrylic acid copolymer LD, and HPC.
If these coating agents and masking agents are liquid, they can be used as they are. Moreover, you may dilute with diluents, such as water. Moreover, if it is solid, what was melt | dissolved or suspended in solvents, such as water, is used.
[0028]
Furthermore, a microcapsule can be formed by spraying a wall film substance from one liquid flow path of a four flow path nozzle instead of a coating agent or a masking agent.
In this case, the bacterial cell liquid is preferably suspended in a solvent inert to the bacterial cells such as glycerin, medium chain triglyceride or corn oil. This is because, when the solvent of the bacterial cell liquid is water, the survival rate of the bacterial cells may be reduced due to a product accompanying active metabolism of the bacteria.
Examples of the wall membrane material include cellulosic materials such as ethyl cellulose; polymer materials such as polylactic acid, polystyrene, polybutadiene, and styrene-methyl acrylate polymer; or melting points higher than body temperature such as hardened palm oil. Examples thereof include hardened oil.
If these wall membrane materials are liquid, they can be used as they are. Moreover, you may dilute with diluents, such as water. In the case of a solid, a solution dissolved or suspended in a solvent such as water is used.
[0029]
As a method for producing a coated or masked dried microbial cell product according to the present invention, or a method for producing a microcapsule containing a dried microbial cell product, not only the above method but also the above-described bacterial cell liquid and coating agent, masking An example is a method in which an agent or a wall membrane substance is mixed in advance and the mixture is subjected to spray drying according to the present invention.
[0030]
The dried bacterial cell product obtained by the above-described spray drying according to the present invention is a compressed gas sent to the nozzle in consideration of whether the bacterial cell liquid is a solution or a suspension, or its properties such as concentration and viscosity. By adjusting the gas amount and the liquid amount of the bacterial cell liquid, a dried bacterial cell product having an arbitrary particle size can be obtained.
However, as described above, the particle size of the dried bacterial cell product is preferably about 1 to 10 μm rounded off to the first decimal place. This is because, as the particle size is smaller, for example, static electricity is less likely to occur in the preparation process or less pressure is applied to the cells during tableting. The particle size of the dried microbial cell can be easily measured with a speculum.
In addition, in order to reduce the particle size of the obtained dried microbial cell product, a known treatment may be performed before spray drying. Known treatments include treatments such as reducing the viscosity and surface tension of the cell fluid, or reducing the primary particle size or suspending well when the cell fluid is a cell suspension. .
[0031]
By reducing the particle size of the dried microbial cell as described above, there is also an advantage that the viable cell rate can be increased and a medicine or food having a large number of viable cells can be provided. That is, in order to obtain a single-micron dried bacterial cell product, the spray droplets are preferably single-micron. When the particle size of the spray droplets is small, the surface area per unit weight of the spray droplets increases, so that contact with the drying air is performed efficiently and drying is performed efficiently even when the temperature of the drying air is relatively low. Therefore, it is possible to suppress the death or damage of the cells due to the heat of the dry wind as much as possible. As a result, the viable cell rate is increased and a dried cell body having a large number of viable cells is obtained.
Therefore, according to the spray drying according to the present invention, the viable cell rate is about 1.0 × 10 ⁵ ~ About 2.0 × 10 ¹² About CFU / g, preferably about 1.0 × 10 ¹¹ ~ 2.0 × 10 ¹² About CFU / g, more preferably about 1.2 × 10 ¹¹ ~ 1.5 × 10 ¹² About CFU / g, most preferably about 1.5 × 10 ¹¹ ~ 8.0 × 10 ¹¹ A dried microbial cell product of about CFU / g can be obtained. Here, the measurement of the number of viable cells in the dried microbial cells varies depending on the microbial cells, but can be easily measured by, for example, the method for quantifying each microbial cell described in the Japanese Pharmacopoeia Standards for Drugs.
[0032]
In the present invention, the spray drying and fluidized bed granulation may be combined. That is, using a device characterized in that the step of performing fluidized bed granulation using a powder and a binder and the drying step of obtaining a single micron dried cell body by spray drying are performed in the same container. In this apparatus, granulation, spray drying of the bacterial cells, and mixing may be performed continuously.
Conventionally, the steps of spray drying, granulation, and mixing have been performed using separate devices, but by performing granulation, spray drying and mixing of bacterial cells in the same device, the manufacturing process can be performed. At the same time, there is an advantage that the mixing uniformity of the dried microbial cell product and the granulated product is improved and classification is less likely to occur.
[0033]
Specific embodiments of the apparatus according to the present invention include a binder spray nozzle capable of spraying a binder from the top to the bottom of the drying chamber, and a fungus capable of spraying a cell fluid from the top to the bottom of the drying chamber. A fluidized bed is installed at the bottom of the device, which is equipped with a body fluid spray nozzle and can keep the powdery material in a fluid state by blowing compressed gas or sucking air in the device with an exhaust fan. Examples include an attached device. Here, the air forming the fluidized bed may be air dried using a known dehumidifier.
Here, in this apparatus, when two spray nozzles are provided and each is used as a binder spray nozzle or a fungus fluid spray nozzle, the spray nozzle is combined with only one spray nozzle. You may make it use together as a nozzle for agent spraying and a nozzle for fungus body fluid spraying.
[0034]
Using the above apparatus, the process of performing fluidized bed granulation using a powder and a binder and the drying process of obtaining a single micron dried product by spray drying are performed in the same apparatus. Specific embodiments of the method for producing the bacterial cell composition will be described below. In the following embodiments, the fluidized bed granulation step is first performed and then the drying step is performed, but these steps may be performed simultaneously.
First, fluidized bed granulation is performed using a powder and a binder. Specifically, (a) by blowing an inert gas such as compressed gas, preferably nitrogen gas, carbon dioxide gas, or the like from the bottom of the device, or (b) sucking air in the device with an exhaust fan, While maintaining the fluid in a fluid state, the binder is sprayed from the nozzle for spraying the binder, and the droplets of the binder are brought into contact with the fluidized powder, thereby agglomerating the powder and granulating the powder. Do.
Here, as the nozzle for spraying the binder, a known one may be used, but it is preferable to use the four-channel nozzle. If a four-channel nozzle is used, since the spray droplets of the binder are reduced, the particle size of the granulated product is also reduced. As a result, the time required for drying the cast product is shortened, leading to an increase in production efficiency. In addition, if a four-channel nozzle is used, a granulated product having a relatively uniform particle diameter can be obtained. As a result, the pulverization and sizing process can be omitted, and classification is difficult to occur when the dried microbial cell product and the granulated product are mixed.
In addition, the particle diameter of the granulated product is preferably about 100 to 500 μm.
[0035]
Next, the bacterial cell liquid is sprayed from the nozzle for spraying bacterial cell liquid, and a single micron dried cell body is obtained by spray drying. At this time, the compressed gas blown from the bottom of the apparatus or the gas in the apparatus sucked by the exhaust fan (hereinafter collectively referred to as “flowing gas”) becomes dry air, and the bacterial liquid At the same time as the spray droplets are dried, the sprayed droplets are mixed with the granulated material that is in a fluid state by the fluidizing gas. As an aspect of the obtained mixture, the granulated particles and the dried microbial particles are independent and, of course, the powder particles and the dried microbial particles are the binder. In the case of forming one particle by the method, there may be mentioned a case where the particle and the granulated particle or / and the dried bacterial cell particle are mixed. In this way, it is possible to easily produce a bacterial cell composition characterized in that a dried product of single-micron bacterial cells is adhered to a granulated product composed of a powdery material and a binder.
Here, the bacterial cell spray nozzle may be a known one, but it is preferable to use the four-channel nozzle. This is because if the 4-channel nozzle is used, the advantages of the 4-channel nozzle in the spray drying described above can also be exhibited in this manufacturing method.
[0036]
One embodiment of the above-described apparatus and a method for producing a bacterial cell composition according to the present invention will be described with reference to FIG.
For fluidized bed granulation, exhausting while spraying the binder 45 from the binder spraying nozzle 47 in a state where the powdery body 48 is accommodated in the apparatus main body 41 according to the present invention having the fluidized bed 42. The fan is driven, and the flowing gas 49 heated by the heat exchanger is continuously introduced from the lower side of the apparatus main body 41. At this time, the flow gas 49 may be dried in advance using a dehumidifier (not shown). The introduced flow gas 49 is sucked upward by the exhaust fan. As the flow velocity of the flow gas 49 increases, the powder 48 is blown up from the fluidized bed 42 and suspended in the air to form a so-called fluidized bed. Then, by bringing the droplets of the binder 45 into contact with the flowing powdery body 48, the powdery body 48 can be aggregated and granulated.
As for spray drying, when the bacterial suspension 44 is sprayed using the nozzle 46 for spraying the bacterial cell liquid, the spray droplets of the bacterial cell liquid are dried by the flowing gas 49 pulled upward from the fluidized bed 42 by the exhaust fan. Is done. At this time, the temperature of the gas 49 for flow is lowered as occasion demands, and is about 2 to 400 ° C., preferably about 5 to 250 ° C., more preferably about 5 to 150 ° C., more preferably. The temperature is preferably about 20 to 70 ° C. This is to suppress as much as possible the death and damage of the cells by the heat of the flowing gas.
The flow product and the dried bacterial cell product produced as described above are mixed in the apparatus 41 by the flow gas 49.
[0037]
Although there is no limitation in particular as the said powdery material used for fluidized-bed granulation, the thing which can be administered or ingested together with a microbial cell dried material is suitable.
Specifically, for example, substances having other pharmacological actions other than dried bacterial cells, excipients, and other fine powder food materials can be used. These may be known per se, or may be a mixture of two or more.
Examples of the fine powdery food material include cinnamon, ginger, mustard, nutmeg, pepper, saffron, sesame, turmeric, parsley, mint, spices such as celery, onion, garlic, herb dried pulverized products, and these Spices such as curry powder and mixed spices prepared by appropriately mixing spices and herbs; juice powders such as apple, orange, lemon, strawberry, pineapple, grapefruit, grape, melon, lime; coffee, green tea, tea , Oolong tea, cocoa, barley tea and other favorite beverage powders; beef extract, pork extract, chicken extract, scallop extract, clam extract, oyster extract, crab extract, shrimp extract, bonito extract and other animal extract powders; garlic extract, onion extract, celery extract, cabbage Plant extract powders such as kiss and bee extract; powdered dairy products such as skim milk powder, whole milk powder, fermented milk powder; egg yolk powder, whole egg powder; cereal powder, animal and plant protein hydrolysates, salt, amino acids And nucleic acid-based seasoning powders.
[0038]
The binder used in fluidized bed granulation is an additive that increases the binding force between the particles of the powder or between the particles of the powder and the dried cells, and representative examples include water-soluble polymers. Can do. Specific examples include hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose and the like. In addition, polyvinyl pyrrolidone, gelatin, polyvinyl alcohol, starch paste, sugar solution, or the like can also be used as a binder.
A compounding agent may be added to the binder as desired. As the compounding agent, those known per se can be used. For example, natural or synthetic surfactants such as lecithin, quillajasaponin, sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, propylene glycol fatty acid ester; or Examples include gelatin, casein, water-soluble peptide, soybean protein, albumin, gluten and other proteins.
[0039]
The medicine or food containing the dried microbial cell according to the present invention may contain other components as long as it contains the dried microbial cell according to the present invention.
For example, components having other pharmacological actions and other foods may be included, and additives or additives having other desired functions may be included as desired.
[0040]
The dry cell product obtained by spray-drying as described above is a pharmaceutical containing the dry cell product according to the present invention in the form of a powder or granule as it is or in the form of a tablet, pill or troche. Or it can be a food.
When the pharmaceutical or food containing the dried bacterial cell in the present invention takes a powder or granule dosage form, the dried bacterial cell obtained by spray drying may be used as it is, or it is practiced in the art. A process known per se may be performed.
[0041]
When the medicine or food containing the dried microbial cell product in the present invention takes the form of a tablet, the dried microbial cell product obtained by spray drying is compressed by a tableting machine. As the tableting machine, for example, a known machine such as a rotary tableting machine can be used.
Additives commonly used in the art such as usually powdery or granular excipients, binders, disintegrants or antistatic agents are usually added to the dried bacterial cells obtained by spray drying according to the present invention. May be produced by tableting with a tableting machine.
Further, the obtained tablets may be coated or masked with sugar coating, enteric coating or film coating. As the coating agent or masking agent, known ones such as those described above may be used. The coating or masking can be performed by a known method or a method according thereto.
The tablet may be a sustained release tablet. The sustained-release tablet includes a graduated product obtained by dispersing the dried microbial cell product obtained by spray drying in a plastic lattice discharged as it is outside the body, and the dried microbial cell product obtained by spray drying is a sponge-like wax. Examples thereof include a wax matrix dispersed in a lattice, a resinate obtained by adsorbing a dried microbial cell product obtained by spray drying to an ion exchange resin, or a span tab having a plurality of layers having different dissolution and release properties. These sustained-release tablets can be produced by known methods or similar methods.
[0042]
When the medicine or food containing the dried microbial cell product in the present invention takes the form of a pill or troche, it can be produced in the same manner as a tablet. However, in the case of a troche, since it is gradually dissolved or disintegrated in the mouth, no disintegrant is usually added.
Capsules may be prepared by filling capsules with dried bacterial cells obtained by spray drying. As the capsule base material, for example, a known capsule base material such as gelatin may be used. In the case of a soft capsule, a plasticizer such as glycerin or sorbitol may be used. In addition, the dried bacterial cell product obtained by spray drying is compression molded by a method such as tableting, impregnated with oil or the like if necessary, and then coated with a solid oil or fat, and then coated with a capsule base material. May be manufactured. By doing so, even if water permeates through the capsule coating, water does not directly touch the dried microbial cells, so that damage or death of the microbial cells due to water can be prevented.
Further, it may be microencapsulated using polylactic acid or the like according to conventionally known means.
[0043]
The medicine or food containing the dried microbial cell product of the present invention may be in a liquid dosage form such as a pharmaceutically acceptable solubilizer, suspension, syrup, or elixir.
Examples of the diluent used include purified water, vegetable oil, and ethanol. In addition to diluents, additives such as wetting agents, suspending agents, sweetening agents, flavoring agents, fragrances or preservatives may be mixed.
[0044]
The suspension is prepared by a known method so that the dried bacterial cell product obtained by spray drying is added to a solvent such as water or vegetable oil together with the suspension or other desired additives, and the whole is homogeneous. It is preferable to make it cloudy. As the suspending agent, known ones may be used, and examples thereof include gum arabic, carmellose sodium, methylcellulose, sodium alginate and the like.
The syrup is preferably produced by dissolving or suspending the dried microbial cell product obtained by spray drying in a diluent such as purified water together with sucrose and optionally other sugars or sweeteners. It is good also as a dry syrup agent which melt | dissolves or suspends a microbial cell dry substance at the time of use.
The elixir is preferably produced by dissolving the dried microbial cells obtained by spray drying in ethanol with sweetness and aroma together with additives as desired. The ethanol content is preferably about 4 to 40% by weight.
[0045]
You may prepare the pharmaceutical containing the microbial cell dry substance in this invention in the injection for parenterals, such as an aqueous injection, a non-aqueous injection, an aqueous suspension injection, or a non-aqueous suspension injection.
For injections, the dried bacterial cells obtained by spray drying are dissolved in a solvent together with additives, if necessary, and then filtered or suspended in a solvent, then filled into a container, and the container is sealed and sterilized. It is preferable to carry out and manufacture.
Examples of the solvent include aqueous solvents such as distilled water for injection, physiological saline or Ringer's solution; vegetable oils such as propylene glycol, polyethylene glycol or olive oil, or non-aqueous solvents such as alcohols such as ethanol.
Examples of additives include: (a) stabilizers such as ethylenediamine, (b) preservatives such as paraoxybenzoates, benzyl alcohol, chlorobutanol, phenol or cresol, and (c) polyoxyethylene hydrogenated castor oil. Surfactants such as sucrose fatty acid esters, or solubilizers such as lecithin or hydrogenated lecithin, (d) isotonic agents such as sodium chloride, glucose or glycerol, (e) eg phosphates or citrate Buffers such as acid salts, or (f) Suspending agents such as gum arabic, carmellose sodium, methylcellulose, or sodium alginate.
[0046]
The medicine or food containing the dried microbial cell product according to the present invention is used for promoting the health of mammals. It can also be used as an agrochemical or as an additive for feed.
For example, lactic acid bacteria are widely known as one of useful intestinal bacteria and are said to have a deep relationship with health. There are many reports on the physiological significance of Bifidobacteria, Lactococci or Lactobacillus, etc., and it suppresses the growth of harmful bacteria by producing organic acids such as lactic acid or acetic acid in the intestine, vitamin production or immunity The activation of has been clarified. Therefore, many products such as pharmaceuticals or foods containing lactic acid bacteria have been developed for the purpose of maintaining and strengthening the health of humans, domestic animals, pets and the like, and preventing or treating diseases.
In addition, lactic acid bacteria are used in the manufacturing process of foods such as miso, soy sauce, pickles, and sake, and are used because they are deeply involved in improving flavor and shelf life or maintaining quality.
[0047]
The medicament containing lactic acid bacteria or bifidobacteria in the present invention is, for example, treatment or prevention of diarrhea, constipation, hypercholesterolemia, liver disease, immunosuppressive disease, enteritis (for example, gastroenteritis or colitis); Intestinal recovery after a malfunction with food, drugs, chemicals or physical agents, or after surgical invasion, chemotherapy or contact infection; used to inhibit endotoxin absorption and antagonize endogenous toxin production be able to.
This is because the medicine has the effect of restoring the intestinal mucosa and regulating them when the enzyme potential of the intestinal tract changes as a result of the stress situation. It also normalizes the function of the intestinal flora, promotes vitamin and protein synthesis, digestion process or enzyme process and absorption process, prevents colonization of pathogenic microorganisms, and stimulates the immune response is there.
[0048]
The use as a food containing the lactic acid bacterium or the bifidobacteria dried product of the present invention is applied to the dried microbial product obtained by the above-mentioned spray drying using a dairy product such as yogurt or cheese, confectionery or beverage. And the like.
Moreover, as a seasoning, the microbial cell dried material obtained by the said spray drying can also be used as it is. Moreover, you may use as a seasoning by dissolving or suspending the dried cell body obtained by the said spray drying in solvents, such as water or oil. Furthermore, you may mix with another seasoning.
[0049]
The dried microbial cell product obtained by spray drying can also be used as a starter for stably producing a lactic acid fermented food on an industrial scale.
Powdered lactic acid bacteria starters are used for dairy products, rye bread, fermented sausages, and the like.
[0050]
The yeast cell product obtained by spray drying can be used in the liquor industry such as beer, sake or wine by applying the property that yeast actively performs alcohol fermentation. It can also be used for fermentation such as bread.
Furthermore, yeast extract, which is a yeast autolysate or hot water extract, contains a large amount of vitamins, organic bases or amino acids and has a high nutritional value. The dried product can be used as a medicine such as a vitamin or a health food.
[0051]
The dried cell product of Aspergillus obtained by spray drying can be used for producing sake, amazake, shochu, miso, mirin or soy sauce. Aspergillus oryzae is often used, but Asp. Nigar is sometimes used for the production of alcohol.
In addition, gonococcus has many strains that produce amylase, protease and other various enzymes, and various organic base vitamins. Accordingly, the dried gonococcal microbial cells in the present invention are used as pharmaceuticals or health foods such as digestive enzymes and vitamins. Can be used.
[0052]
【Example】
[Example 1]
[Culture of bacterial cells]
0.1% by weight of a Bifidobacterium bifidum culture solution enriched in advance in a GAM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was inoculated into the medium and cultured at 37 ° C. for 16 hours. This was centrifuged, and the resulting precipitate was used as wet cells.
[0053]
[Creation of bacterial suspension]
35% by weight of dextrin in an aqueous solution adjusted to a concentration of 0.5M mannitol, 0.5M sodium glutamate, 0.1M arginine, 0.1M sodium succinate and 0.01M magnesium sulfate The obtained wet cells were suspended in a dispersion medium prepared by adding so that
[0054]
[Measurement of viable count in bacterial suspension]
The measurement was carried out according to the bifidobacteria quantitative method described in the section of the Japanese Pharmacopoeia Pharmaceutical Standards “Bifidobacteria”. That is, 1 mL of the bacterial suspension was taken and diluted so that the number of viable bacteria in 1 mL was 20 to 200 by a 10-fold dilution method using a diluent prepared as described below. 1 mL of this solution was placed in a Petri dish, and 20 mL of an agar medium for bifidobacteria test maintained at 50 ° C. was added thereto and quickly mixed to solidify. This was anaerobically cultured at 37 ° C. for 60 hours, and the number of viable bacteria in the bacterial suspension was determined from the number of colonies that appeared and the dilution factor.
[0055]
Here, the agar medium for Bifidobacterium test was heated to dissolve components other than horse blood among the following components, adjusted to pH 7.2, and heated at 115 ° C. for 20 minutes using a high-pressure steam sterilizer. After sterilization, it was cooled to 50 ° C. and added with horse blood.
150 mL of bovine liver leachate
10g peptone made of beef
Casein peptone 5g
Yeast extract 5g
3g meat extract
Soybean peptone 3g
Glucose 10g
Potassium monohydrogen phosphate 1g
1g potassium dihydrogen phosphate
0.5g starch
L-cysteine hydrochloride 0.5g
Magnesium sulfate 0.2g
Sodium chloride 0.01g
Ferrous sulfate 0.01g
Manganese sulfate 7mg
Polysorbate 80 1g
Kanten 15g
Horse blood 50mL
790 mL of purified water
pH 7.1-7.3
[0056]
The diluent was prepared by mixing the following components and sterilizing by heating at 121 ° C. for 15 minutes using a high-pressure steam sterilizer.
Anhydrous sodium monohydrogen phosphate 6.0 g
4.5g potassium dihydrogen phosphate
Polysorbate 80 0.5g
L-cysteine hydrochloride 0.5g
Kang 1.0g
Purified water 1000mL
pH 6.8-7.0
[0057]
[Spray drying]
The bacterial suspension prepared as described above was spray-dried using the spray dryer shown in FIG. 2 incorporating a four-channel nozzle (described in Japanese Patent No. 2797080). That is, the bacterial suspension is supplied from two liquid channels at a rate of 8 mL / min (4 ml / min / 1 mm nozzle), and compressed gas is converted to 40 NL / min (converted at 20 ° C.) from the two gas channels. Spray droplets generated by supplying at a rate of (value) were dried with a drying wind to produce a dried bacterial cell product according to the present invention. The drying air has an inlet temperature of 100 ° C. and 1.0 m. ³ It was supplied into the drying chamber at a rate of / min and the outlet temperature was 67 ° C.
[0058]
[Measurement of the number of viable bacteria in dried bacterial cells]
The number of viable cells in the obtained dried bacterial cells was measured according to the bifidobacterial quantification method described in the section of the Japanese Pharmacopoeia Pharmaceutical Standards “Bifidobacteria”. That is, 5 g of the dried microbial cell product was taken up in the same diluted solution previously heated to 37 ° C., and the total amount was 50 mL. This was stirred well and heated at 37 ° C. for 30 minutes to obtain a sample stock solution. This was diluted so that the number of viable bacteria in 1 mL was 20 to 200 by a 10-fold dilution method using a diluent. 1 mL of this solution was placed in a Petri dish, and 20 mL of an agar medium for bifidobacteria test maintained at 50 ° C. was added thereto and quickly mixed to solidify. This was anaerobically cultured at 37 ° C. for 60 hours, and the number of viable bacteria in the dried microbial cell was determined from the number of colonies that appeared and the dilution factor.
[0059]
It implemented similarly by making the entrance temperature of dry air into 80 degreeC, 60 degreeC, and 50 degreeC sequentially.
[0060]
[Comparative Example 1]
The bacterial suspension obtained in the same manner as in Example was lyophilized. The lyophilization conditions were a shelf temperature of 30 ° C. or lower and an ultimate vacuum of 0.1 Torr (13.3 Pa).
The number of viable bacteria in the powder after drying was measured in the same manner as in Example 1.
[0061]
Table 1 shows the number of viable bacteria and the survival rate of Examples and Comparative Examples.
Here, the survival rate was calculated from the following equation.
Survival rate (%) = {(number of viable cells per gram of dried microbial cell × solid content of bacterial suspension) / (number of viable cells per gram of bacterial suspension)} × 100
[Table 1]

As shown in Table 1, it was found that when the spray drying according to the present invention is used, a dried microbial cell product having a higher survival rate than that obtained by lyophilization can be obtained.
[0062]
[Particle size of dried bacterial cell]
In Example 1, the dried bacterial cell product according to the present invention obtained at an inlet temperature of 60 ° C. was measured using a microscope (manufactured by Keyence Corporation, Digital HD Microscope VH-7000). The particle size distribution was as follows.
[Table 2]

[0063]
[Example 2]
In the same manner as in Example 1, the cells were cultured, a cell suspension was prepared, and the number of viable cells in the cell suspension was measured.
(Spray granulation)
In the fluidized bed granulator FLO-120 (made by Freund Corporation), in addition to the conventional two-channel spray nozzle, an apparatus further incorporating a four-channel nozzle (described in Japanese Patent No. 2797080) A powder containing lactic acid bacteria was prepared by spraying a suspension of lactic acid bacteria.
The process was roughly divided into two, and in the first process, a binder was sprayed on the excipient as a granular material using a two-channel nozzle to perform granulation. In the second step, the lactic acid bacteria suspension was spray-dried using a four-channel nozzle while the fluidized bed was maintained at a low temperature, and simultaneously mixed with the particulate matter obtained in the first step.
The operating condition of the machine used in this experiment is an air supply volume of 70 m. ³ Nominal air amount was 40 NL / min (value converted at 20 ° C.).
Moreover, the composition of the excipient | filler and binder used for granulation used the following.

[0064]
The first step will be described in detail below. 120 kg of the above-mentioned shaping raw material is weighed and put into a hopper, the supply air temperature is set to 100 ° C., dry air is fed to form a fluidized bed, and the binder is sprayed at a liquid flow rate of 1.6 L / min using a two-channel nozzle. did. The exhaust temperature at this time was 45-50 degreeC, and the temperature in a fluidized bed was 55-60 degreeC. When the spraying of the binder was completed, it was directly dried in a fluidized bed for 20 minutes to obtain a low moisture granulated product.
[0065]
Next, the second step will be described in detail below. After completion of the post-drying of the granulated product, 350 mL of the bacterial suspension prepared in the same manner as in Example 1 was sprayed at a liquid flow rate of 16 mL / min using a 4-channel nozzle with the supply air temperature lowered to 70 ° C. . The exhaust temperature at this time was 30-35 degreeC, and the temperature in a fluidized bed was 35-40 degreeC. Lactic acid bacteria were pulverized by spray drying at such a low temperature, and at the same time, the lactic acid bacteria-containing powder according to the present invention was obtained by mixing in a fluidized bed.
[0066]
[Comparative Example 2]
A mixed powder of lactic acid bacteria was prepared so that the same amount of lactic acid bacteria was contained in the preparation.
2 g of the microbial cell powder obtained in Example 1 was doubled with corn starch to 20 g, and 1180 g of a granulated product consisting of the excipient and binder obtained in the first step of Example 2 was used. In addition, a mixture of 100 rpm for 3 minutes with a V-type mixer was used as a mixed powder of lactic acid bacteria.
[0067]
The number of viable bacteria contained in 1 g of the powder obtained in Example 2 or Comparative Example 2 was measured according to the section of [Measurement of the number of viable bacteria in the dried bacterial cell] of Example 1. In Example 2, the number of viable bacteria was 5.12 × 10 ⁸ (CFU / g), and in the comparative example, the viable cell count was 4.85 × 10 ⁸ (CFU / g).
Therefore, by using the method according to the present invention which combines spray drying and fluidized bed granulation, the viable cell rate is at least as high as or more than that of the conventional powder production method comprising spray drying, granulation and mixing steps. It has been found that the manufacturing process can be simplified while keeping it.
[0068]
Example 3
The following components were added to 500 g of distilled water and stirred to prepare a coating agent.

On the other hand, a bacterial suspension was prepared in the same manner as in Example 1.
It spray-dried using the spray-dryer shown in FIG. 2 incorporating the 4 flow-path nozzle (the thing of patent 2797080 description). That is, a bacterial cell liquid is supplied at a rate of 5 g / min from one liquid channel in a four-channel nozzle having two liquid channels and two gas channels, and a liquid coating agent is supplied from another liquid channel. Is sprayed at a rate of 20 g / min, and compressed droplets are supplied from two gas flow paths at a rate of 40 NL / min. A dried product was produced.
The dry air has an inlet temperature of 100 ° C. and 1.0 m ³ It was supplied into a drying chamber of 0.9 mPa at a rate of / min.
When 10 mg each of the conventional bifidobacteria raw powder and the partially coated microbial cell dried product according to this example were taken and a taste test was performed, the taste of the partially coated microbial cell dried product according to this example was improved.
[0069]
Example 4
100 g of the bacterial suspension prepared in the same manner as in Example 1 and an enteric coating base having the following composition were mixed, and the mixed solution was shown in FIG. 2 incorporating a four-channel nozzle (described in Japanese Patent No. 2797080). Spray drying was performed using a spray dryer. That is, the mixed liquid is supplied from the two liquid channels at a rate of 5.6 g / min, and the spray droplets generated by supplying the compressed gas from the two gas channels at a rate of 40 NL / min are dried. It dried with the wind and manufactured the enteric-coated dry cell body concerning this invention.
The dry air has an inlet temperature of 80 ° C. and 1.0 m ³ It was supplied into a drying chamber of 0.9 mPa at a rate of / min.

[0070]
[Acid resistance test of enteric-coated dried bacterial cells]
An acid resistance test was performed using the first solution (pH 1.2, 36 to 38 ° C.) described in Japanese Pharmacopoeia, General Test Method, Disintegration Test Method. The first liquid was put in a container capable of keeping about 1000 mL and kept at 36 to 38 ° C. The resulting dried microbial cell product was added and stirred well, and 1 mL each was sampled 5, 15, 30, 60 minutes after the dried microbial cell product was added. The sampled donations were diluted in order to the appropriate concentration by adding the diluent described in Example 1, and the number of viable bacteria was measured in the same manner as in Example 1. The first pharmacopoeia liquid is an artificial gastric juice that is dissolved in 2.0 g of sodium chloride by adding 7 mL of hydrochloric acid and distilled water to make 1000 mL.
Moreover, the same test was done also about the conventional Bifidobacterium bulk powder.
FIG. 3 shows the relationship between the first solution protection time and the number of viable bacteria. In the conventional fungal powder, the number of viable bacteria becomes 1 / 1,000 or less after 5 minutes, and no viable bacteria are observed after 30 minutes, whereas in the coated fungal powder according to the present invention, Even after 60 minutes from the start, about 1% of viable bacteria were observed. That is, the acid resistance of the dried microbial cells was greatly improved by performing the above coating.
[0071]
Example 5
A bacterial suspension was prepared in the same manner as in Example 1 except that Lactobacillus acidophilus was used as the bacterial cell.
The number of viable bacteria in the bacterial suspension was measured according to the determination method of lactamiton described in the section of the Japanese Pharmacopoeia Pharmaceutical Standards “Lacmitone”. That is, 1 ml of a bacterial suspension was taken and diluted with a diluted solution prepared as described below so that the number of viable bacteria in 1 ml was 20 to 200 by a 10-fold dilution method. 1 ml of this solution was placed in a Petri dish, and 20 ml of a lactene test agar medium maintained at 50 ° C. was added thereto and quickly mixed to solidify. This was cultured at 37 ° C. for 48 hours, and the number of viable bacteria in the bacterial suspension was determined from the number of colonies that appeared and the dilution factor.
[0072]
Here, an agar medium for lamitone test was prepared by mixing the following components and sterilizing by heating at 121 ° C. for 15 minutes using a high-pressure steam sterilizer.
Here, the tomato juice was prepared by adding an equal amount of purified water to the tomato juice and boiled with occasional stirring, then adjusting the pH to 6.8 and filtering.
Casein peptone 20g
Yeast extract 5g
Meat extract 15g
Glucose 20g
L-cysteine hydrochloride 1g
Polysorbate 80 3g
200ml tomato juice
Kanten 20g
800 mL of purified water
pH 6.8
[0073]
The diluent was prepared by mixing the following components and sterilizing by heating at 121 ° C. for 15 minutes using a high-pressure steam sterilizer.
Sodium monohydrogen phosphate 6.0g
4.5g potassium dihydrogen phosphate
Polysorbate 80 0.5g
L-cysteine hydrochloride 0.5g
Kang 1.0g
1000 ml of purified water
pH 6.9
[0074]
The bacterial suspension prepared as described above was spray-dried in the same manner as in Example 1.
The machine operating conditions were the same as in Example 1 except that the inlet temperature was 60 ° C and the exhaust temperature was 45 ° C.
The number of viable cells in the obtained dried bacterial cell was measured according to the method for quantitative determination of lactamitone described in the Japanese Pharmacopoeia Standards for Drugs “Lacmitone”. That is, 5 g of the dried microbial cell product was taken up in a diluted solution preliminarily heated to 37 ° C. to make the total volume 50 mL. This was stirred well and heated at 37 ° C. for 30 minutes to obtain a sample stock solution. This was diluted so that the number of viable bacteria in 1 mL was 20 to 200 by a 10-fold dilution method using a diluent. 1 mL of this solution was placed in a Petri dish, and 20 mL of agar medium for lacriton test kept at 50 ° C. was added thereto and quickly mixed to solidify. This was anaerobically cultured at 37 ° C. for 48 hours, and the number of viable bacteria in the dried microbial cell was determined from the number of colonies that appeared and the dilution factor.
[0075]
The number of viable cells in the dried bacterial cell is 7.24 × 10 ¹¹ (CFU / g) and the survival rate was 90%. The survival rate was determined in the same manner as in Example 1.
Therefore, it was found that by using the spray drying according to the present invention, the survival rate of the bacterial cells was increased, and as a result, a dried bacterial cell product having a very large number of viable cells was obtained.
[0076]
【The invention's effect】
By using a spray drying apparatus equipped with a atomizing apparatus capable of forming single micron spray droplets, the surface area per unit weight of the spray droplets is increased, and contact with the drying air is efficiently performed. Therefore, when drying is performed with drying air at a temperature similar to that of conventional spray drying, the time required for drying the bacterial cell liquid is shortened, and the death or damage of the bacterial cells can be suppressed as much as possible. Even if the temperature of the drying air is lower than the temperature of the drying air in the conventional spray drying, sufficient drying can be performed without reducing the yield or stability, and the temperature of the drying air is low. The death or damage of the bacterial cells due to heat can be suppressed as much as possible. Therefore, by the method for producing a dried microbial cell product according to the present invention, a dried microbial cell product having a large number of viable cells can be obtained.
[0077]
In addition, dried bacterial cells containing single-micron-sized particles obtained by drying spray droplets having a small particle diameter with drying air have a low pressure on the bacterial cells during compression molding or the like. It is easy to mold into pharmaceuticals or foods in dosage forms such as tablets.
[0078]
As the spray drying apparatus according to the present invention, by using a spray drying apparatus having a four-channel nozzle having two liquid channels and two gas channels, a large amount of spraying is possible because there are two liquid channels. In addition to improving production efficiency, two different solutions or suspensions can be sprayed simultaneously from separate liquid channels to produce a dried microbial cell mixture in which these are mixed.
Further, by spraying a liquid masking agent or coating agent from one liquid flow path and a bacterial cell liquid from the other liquid flow path, it is possible to produce a masked or coated dry cell body. Therefore, it is not necessary to perform masking or coating separately, and the manufacturing process is reduced. Similarly, microcapsules can also be produced by spraying the capsule substrate simultaneously with the bacterial cell liquid.
Furthermore, since the particle size distribution is within a relatively narrow range by using a spray drying apparatus having a four-channel nozzle, it is easy to perform subsequent steps such as granulation and sieving.
[0079]
Spray drying is useful both in terms of cost and resources because the scale of the apparatus is smaller than that of freeze drying and less energy is required to operate the apparatus.
Further, the step of performing fluidized bed granulation using the powder and the binder according to the present invention and the step of obtaining a single micron dried cell product by spray drying are performed in the same container. Using the device, granulation, spray drying and mixing of the bacterial cells are continuously performed in the device, so that the manufacturing process can be simplified and at the same time the mixing uniformity of the dried bacterial cell product and the granulated product is improved. Classifying is less likely to occur.
[Brief description of the drawings]
FIG. 1 shows an internal structure of a nozzle edge portion in a spray drying apparatus having four flow path nozzles.
FIG. 2 shows a schematic diagram of a spray drying apparatus having a four-channel nozzle.
FIG. 3 shows the change over time in the number of viable bacteria in an acid resistance test of a dried enteric-coated bacterial cell.
FIG. 4 is characterized in that the step of performing fluidized bed granulation using the powder and the binder according to the present invention and the step of obtaining a single micron dried product by spray drying are performed in the same container. A schematic diagram of the apparatus is shown.
[Explanation of symbols]
1, 2 Gas flow path to which compressed gas is supplied
3, 4 Liquid channel through which a liquid containing a material to be dried is supplied
5 Fluid flow surface
6 Collision focus
7 Spray droplets
21 Bacterial fluid, coating agent or masking agent
22 Liquid flow path
23 Gas flow path
24 Compressed air inlet
25 Dry air inlet
26 Entrance thermometer
27 Atomizer
28 Spray nozzle
29 Outlet thermometer
30 Drying chamber pressure gauge
31 Drying room
32 Dried cell exit
33 Rectifier
41 Main unit
42 Fluidized bed
43 Bug Filter
44 Bacterial fluid
45 binders
46 Nozzle for spraying bacterial cells
47 Binder spray nozzle
48 powder
49 Gas for flow

Claims (7)

Cell dry matter average particle size is a single micron. The viable cell count is 1.0 × 10 ^{5 to} 2.0 × 10 ¹² CFU / g.  The bacterial cells are bifidobacteria, lactobacilli, lactic acid cocci, spore-forming lactic acid bacteria, saccharifying bacteria, yeast, bacilli, actinomycetes, Bacillus toyoi, B. The dried cell product according to claim 1 or 2, which is licheniformis, butyric acid bacteria, acetic acid bacteria or amino acid fermenting bacteria.  The dried bacterial cell product according to any one of claims 1 to 3, further coated or masked. A microbial cell composition , wherein a microbial cell dry product having an average particle size of single micron is attached to a granulated product comprising a powdery material and a binder.  The pharmaceutical or foodstuff containing the microbial cell dried material of Claims 1-4, or the microbial cell composition of Claim 5.  It is a tablet, The pharmaceutical or foodstuff of Claim 6 characterized by the above-mentioned.

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